Concerns that Francisella tularensis, the bacterial agent of tularemia, might be exploited as a weapon of bioterrorism have led to greatly expanded research efforts to understand the basis of its pathogenicity. One factor contributing to virulence of F. tularensis in mammals is its ability to replicate within host cells. Although interactions with macrophages have been best studied, the bacterium also grows to high numbers in hepatocytes, and,indeed, the liver is a major target of infection. Within host cells, F. tularensis is shielded from the immune response. Moreover, F. tularensis incites only a limited array of pro-inflammatory changes in endothelial cells, implying that endothelial recruitment of leukocytes to infected tissues is impaired. We therefore hypothesize that F. tularensis interacts with host cells and the innate immune system in an exceptional manner that contributes to the high infectivity and virulence of the organism. Three specific aims are proposed to explore this premise: 1) To determine whether virulence of F. tularensis strains correlates with their ability to replicate within, kill, or alter expression of specific genes in host cells. There are relatively few studies exploring interactions of virulent strains of F. tularensis with primary cultures of human cells. Consequently, there is little knowledge regarding the degree to which virulence of the bacterium is linked to its effects on specific human cells. To fill this gap,strains of F. tularensis of varying virulence will be characterized with respect to their interactions with human hepatocytes, endothelial cells, and leukocytes using DMA microarrays and other standard methodologies. 2) To elucidate the mechanisms by which F. tularensis suppresses pro-inflammatory activation of endothelial cells. Preliminary data indicate that the poor response of endothelial cells to F. tularensis is due to active suppression of pro-inflammatory pathways by the bacteria. Through in vitro approaches, the means by which F. tularensis limits the response of endothelium will be defined. 3) To characterize the role of the hepatocyte in the pathogenesis of tularemia. Despite the ability of F. tularensis to replicate within hepatocytes, the contribution of these host cells to development of disease is an unexplored topic. This aim will identify components of the bacterium that support its growth in hepatocytes, as well as hepatocytic products that are likely to initiate and propagate an innate immune response to F. tularensis in the liver. Results of Aim 1 will help guide experiments proposed in Projects 3, 5, and 6 of this Program Project Grant. Aims 2 and 3 will be carried out in collaboration with Drs. Erich Mackow and Jorge Benach, the leaders of Project 4 and Core B, respectively. RELEVANCE (Seeinstructions): There is concern that Francisella tularensis could be exploited as a biological weapon due to its high infectivity and capacity to cause lethal disease in humans. The proposed research will help in understanding how this bacterium avoids the normal antimicrobial defenses of the body and will provide a basis for developing new strategies for treatment should F. tularensis be released in an act of terrorism.